This proposal focuses on the effects of peroxynitrite on neutrophil functions. Based on the observations by these investigators that peroxynitrite could serve a as priming agent for neutrophil functions, and that this effect may be mediated primarily by nitration of tryrosine residues on neutrophil proteins, they propose that neutrophil function is modulated during inflammation or other periods of oxidant stress by tyrosine nitration on specific neutrophil proteins. To investigate this hypothesis, they propose to 1) analyze the effects of peroxynitrite-mediated tyrosine nitration on neutrophil functional responses such as calcium flux, rolling, adhesion, chemotaxis, degranulation, phagocytosis, and cytoine production; 2) analyze the effects of peroxynitrite treatment of purified NADPH oxidase proteins on oxidase assembly and enzymatic activity in vitro; 3) identify neutrophil proteins that are modified by tyrosine nitration in intact cells by immunoprecipitation, Western blotting, and amino acid sequencing; and 4) map specific sites of nitration on neutrophil proteins using mass spectrometry, peptide mapping, and amino acid sequencing.